DNA sequences encoding proteins used to elicit and detect programmed cell death

ABSTRACT

Polypeptides and mutants and variants associated with programmed cell death in mammalian cells and DNA sequences, and fragments and derivatives thereof, encoding the polypeptides are disclosed. Also disclosed are methods for detecting programmed cell death in mammalian cells, a method of activating programmed cell death in unwanted mammalian cells, and methods for preventing unwanted cell death occurring in degenerative disorders of mammals.

The invention described herein was made in the course of work under agrant or award from The Department of Health, Education and Welfare andthe Government has certain rights therein.

This is a division of application Ser. No. 07/915,934, filed Jul. 20,1992now U.S. Pat. No. 5,360,893.

FIELD OF THE INVENTION

The present invention relates to the diagnostic and therapeutic uses ofthe first cloned sequences of genes associated with programmed celldeath (PCD) in mammalian cells. More specifically, the invention relatesto the programmed cell death genes of RP-8 and RP-2.

BACKGROUND OF THE INVENTION

"Programmed" as opposed to "accidental" death of cells is a normal andessential biological feature in the differentiation and maintenance ofcellular populations in multicellular organisms. The normal turnover ofepithelia such as skin or the gut lining involves the programmed deathof terminally differentiated cells. Similarly, many cells of thehematopoietic system have short life expectancies, and their death isalso programmed. In the developing nervous system, large numbers ofneurons undergo programmed cell death (Oppenheim, Trends Neurosci.8:487-493 (1985)).

Programmed death can often be distinguished morphologically fromaccidental death (Wyllie, Int. Rev. Cytol. 17:755-785 (1987); Wyllie, etal., Int. Rev. Cytol. 68:251-300 (1980)). In accidental death, the majortarget organelle seems to be the mitochondrion, which swells until it isdysfunctional, leading to death and lysis of the cell (necrosis). Incontrast, programmed death is usually characterized by an early collapseof the nucleus, with extreme condensation of chromatin and loss of thenucleolus. The cell nucleus, with extreme condensation of chromatin andloss of the nucleolus. The cell shrinks, in contrast to swelling innecrosis, and is phagocytosed before it lyses. This phenomenon, firstreferred to as shrinkage necrosis, is now called apoptosis (Kerr, etal., Br. J. Cancer 26:239-257 (1972)). The nuclear collapse in apoptosisis probably due, in most cell types, to fragmentation of the chromatininto units of single or multiple nucleosomes observable byelectrophoresis in agarose gels (Cohen and Duke, J. Immunol. 132:38-42(1984); Wyllie, Nature 284:555-556 (1980)). This is one of the ways inwhich apoptosis can be distinguished from necrosis. Chromatinfragmentation may be the result of activation of an endogenous Ca²⁺ andMg²⁺ -dependent endonuclease (Compton and Cidlowski, J. Biol. Chem.262:8288-8292 (1987); Hewish, Biophys. Res. Commun. 52:475-481 (1973)).

An example of programmed cell death is demonstrated in rodentthymocytes. The death program can be initiated in these cells by anumber of inductive stimuli, including exposure to glucocorticoids(Cohen and Duke, J. Immunol. 132:38-42 (1984); Wyllie, Nature284:555-556 (1980)) and irradiation (Sellins and Cohen, J. Immunol.139:3199-3206 (1987)). In these instances the death program (apoptosis)can be prevented by the presence of inhibitors of RNA or proteinsynthesis (Cohen and Duke, J. Immunol. 132:38-42 (1984), Sellins andCohen, J. Immunol. 139:3199-3206 (1987)). This suggests that genes,normally silent or negatively regulated, are activated by inductivestimuli leading to the production of proteins that mediate or actdirectly in the death process.

SUMMARY OF THE INVENTION

The present invention provides purified polypeptide products and theirmutants and variants associated with programmed cell death in mammaliancells. The particular polypeptides are portions of two previouslyunknown genes, RP-8 and RP-2. The invention also concerns DNA sequences,and fragments and derivatives thereof, encoding these polypeptideproducts, antibodies to the polypeptides, and proteins of thesepreviously unknown genes, and host cells involved in the expression ofthe polypeptide products.

Another aspect of the invention includes a method for detectingprogrammed cell death in various mammalian cells. This involvesgenerating antibodies that react with the polypeptide and proteinproducts of programmed cell death genes, such as RP-8 and RP-2. Anothermethod for detecting programmed cell death is the use of probes thatbind to the RNA of genes associated with programmed cell death ofvarious mammalian cells.

A further aspect of the invention involves a method of activatingprogrammed cell death in unwanted mammalian cells. Included among thesecells are cancer cells and immune cells linked to autoimmune diseases.Cell death activation is accomplished by selectively activating a gene,such as RP-8 or RP-2, that expresses a protein which elicits programmedcell death in the unwanted cell.

An additional aspect of the invention involves a method for preventingunwanted cell death occurring in a degenerative disorder of a mammal.Degenerative disorders known to involve cell death are Alzheimer'sdisease, Parkinson's disease, and Huntington's disease. Compounds usedin preventing unwanted cell death can be antibodies to a proteinassociated with programmed cell death in such disorders or drugsdesigned to inhibit the activity of such a protein. In addition, the useof oligonucleotides complementary to an mRNA associated with programmedcell death can prevent unwanted cell death in such disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic outline of a cloning procedure leading toconstruction of a subtracted library enriched for sequences expressed indeath-induced thymocytes;

FIG. 2 represents a DNA blot showing effectiveness of subtractivehybridization and Taq polymerase (PCR) amplification of subtractedssDNA;

FIG. 3 shows Northern blot analysis of candidate clones, RP-2 and RP-8;

FIG. 4 shows temporal appearance of RP-2 and RP-8 mRNAs in thymocytesfollowing low-dose gamma irradiation;

FIG. 5 (SEQ ID NO:1) represents the nucleotide sequence and amino acidsequence of the sense strand of clone RP-8; and

FIG. 6 (SEQ ID NO:3) represents the nucleotide sequence and amino acidsequence of the sense strand of clone RP-2.

DETAILED DESCRIPTION OF THE INVENTION

A cDNA library is constructed that is enriched for sequences expressedin thymocytes induced to die. To construct this library, a directionalcloning strategy is devised by combining certain recent proceduraladvances that have been used to produce subtracted (enriched) cDNAlibraries (Duguid et al., Proc. Natl. Acad. Sci. USA 85:5738-5742(1988); Palazzolo and Meyerowitz, Gene 32:197-206 (1987); Sive and St.John, Nucleic Acids Res. 16:10937 (1988)). Various types of proceduresfor subtractive hybridization have resulted in the isolation of clonescorresponding to the T-cell receptor (Hedrick et al., Nature 308:149-153(1984)); a muscle-specific regulatory protein, myogenin (Wright et al.,Cell 56:607-617 (1989)); genes expressed in growth-arrested cells(Schneider et al., Cell 54:787-793 (1988)); mRNAs in scrapie-infectedbrains (Duguid et al., Proc. Natl. Acad. Sci. USA 85:5738-5742 (1988));and mRNAs in the cortex of the brain (Travis and Sutcliffe, Proc. Natl.Acad. Sci. USA 85:1696-1700 (1988)). From a library enriched for cDNArepresenting mRNAs specifically present in death-induced cells, clonedcDNAs complementary to mRNAs are isolated that appear soon after cellsare exposed to a stimulus that triggers programmed cell death. ThesemRNAs are considered to be potential "death gene" products since theirpresence is death associated, and not simply single-stimulus associated.

RP-2 and RP-8 are death-associated mRNAs that appear upon induction bystimuli that are known to trigger expression of a death program inthymocytes. RP-2 and RP-8 mRNAs are detected at different timesfollowing induction of death by irradiation, and they both accumulate inthe cytoplasm when the mRNA specifying the abundant protein actin isdeclining.

RP-2 and RP-8 mRNAs are not unique to thymocytes and they appear uponinduction of death in other types of cells, such as epithelial cells andneurons. Expression is shown to be associated with death induced bydifferent stimuli in other types of cells which show that RP-2 and RP-8are involved in programmed cell death.

Both RP-2 and RP-8 mRNAs specify proteins that are essential inprogrammed cell death, as is apparently the case of the products fromced-3 and ced-4 genes in the nematode Caenorhabditis elegans (Yuan andHorwitz, Dev. Biol. 138:33-41 (1990)). In this regard, the zinc fingerprotein encoded by RP-8, which is expressed soon after induction beforemorphological changes are apparent, functions as an essential regulatorycomponent. Therefore, the use of complementary oligonucleotides perturbor block programmed cell death. Such oligonucleotides are readily takenup by mammalian cells in culture, leading to hybridization-basedblockage of translation or the degradation of specific mRNAs.

The RP-2 and RP-8 mRNAs are death associated because they are isolatedfrom a library constructed with mRNA from dexamethasone andcycloheximide-treated thymocytes and they are also shown to be expressedat increased levels in irradiated thymocytes. Since RP-2 and RP-8 mRNAsare consistently expressed in a variety of cell types induced to die bya number of different stimuli, they are involved in the process ofprogrammed cell death.

Construction Of A Library Enriched For Sequences Expressed InDeath-Induced Thymocytes

In order to obtain an enriched or subtracted library, two source cDNAlibraries are first constructed to provide a convenient supply of copyRNA and cloned cDNA molecules. The "programmed" library represents mRNAfrom thymocytes induced (or programmed) to die by exposure todexamethasone in the presence of cycloheximide. The "control" libraryrepresents mRNA from untreated thymocytes. The cDNA libraries areconstructed such that the cDNA inserts in each of the two libraries areoriented in the cloning vector, Lambda Zap, in the same direction(directional cloning). Dying cells present a unique problem in preparingpoly(A)+ mRNA used in the construction of these libraries. Virtuallynothing is known about the number of genes that encode the death programor their temporal expression following exposure to the death stimulus.Since the basic strategy for cloning putative death genes is based upontheir unique or increased expression in dying cells, the time followinginduction when RNA is harvested for library construction couldsignificantly influence screening results. This uncertainty iscompounded further by the fact that in apoptotic cells much of the RNAis degraded (Cidlowski, Endocrinology 111:184-190 (1982)). To have asmany cells as possible in the population committed to the death program,while avoiding the isolation of RNA from cells where accelerateddegradation might be occurring, cycloheximide is added to the culturemedium at the time of induction. Cycloheximide blocks protein synthesisand prevents expression of death processes, while allowing expression atthe nucleic acid level (Sellins and Cohen, J. Immunol. 139:3199-3206(1987)). Thus, mRNA accumulates under these conditions in death-inducedthymocytes. To ensure that the death program is induced at the timecells are harvested for making RNA, a small aliquot of cells is removedfrom the induced culture and washed free of cycloheximide and inducer.These cells are then continued in a subculture; all cells die within 8to 12 h. A second point regarding mRNA preparation pertains to thecontrol, or noninduced, culture of thymocytes. Of thymocytes removedfrom the thymus, about 5 to 10% are in various phases of the deathprocess as they are placed in culture. Thus, in preparing template mRNAfor the cDNA used to produce the control library, as many of the dyingcells are first removed as possible by centrifugation through a Percollgradient (Wyllie and Morris, Am. J. Pathol. 109:78-87 (1982)).

FIG. 1 provides an overview of the strategy that is applied to producethe enriched or subtracted library by using these two cDNA libraries.Directional cloning can be achieved by use of any two of the uniquerestriction endonuclease sites present in the promoter-flanked multiplecloning site of the vector. In this case, a primer-adaptoroligonucleotide is used containing the base sequence recognized by XbaIendonuclease to primer first-strand cDNA synthesis (FIG. 1).

Double-stranded cDNA is obtained by the RNase-H digestion and DNApolymerase fill-in method of Gubler and Hoffman (Gene 25:263-269(1983)). The resultant double-stranded cDNA is blunt ended by using T4polymerase and oligonucleotide linkers containing the EcoRI restrictionsequence are then ligated to the cDNA. To prepare the cDNA for insertioninto Lambda Zap, digestions are performed by using EcoRI and XbaIendonucleases. This cDNA inserts into similarly cut vector so that whenthe recombinant phage is used in helper phage-assisted production ofclosed circular ssDNA from the programmed library, the resultant ssDNAis sense strand (i.e., complementary to cDNA).

Since insertion is directionally controlled rather than random, thecloned sequences in the control library can be transcribed in vitro aseither sense or antisense RNA molecules by using the T3 and T7 promotersites present at opposite sides of the cloning region. As shown in FIG.1, use of T3 polymerase yields antisense RNA, and T7 polymerase, whichtranscribes the opposite strand, produces sense RNA (i.e., equivalent tomRNA).

The control and programmed libraries provide RNA and ssDNA to use forsubtractive hybridization of cDNAs representing mRNAs shared betweencontrol and induced cells (the bulk of the different cloned sequences).Hybridization driven with biotinylated antisense copy RNA from thecontrol library removes about 97% of the sense strand insert-containingssDNA present in the programmed library. See Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Subtractive hybridization with copy RNA                                       ssDNA      Driver RNA    c.sub.0 t                                                                            % Hybridization                               ______________________________________                                        Phagemid-cDNA                                                                            Copy RNA (T3) 1.200  97                                            SK(-)      Copy RNA (T3) 1.700  14                                            SK(-)      Copy RNA (T7) 1.700  12                                            SK(-)      Yeast tRNA    1.700   0                                            ______________________________________                                    

As shown in Table 1, ssDNA (see examples below) from the programmedlibrary was iodinated, and the recombinant (phagemid+inserted cDNA)molecules were isolated by electrophoresis in agarose gels. SK(-), ³H-labeled vector ssDNA. Copy RNA was obtained by in vitro transcriptionof cDNA in the control library. Sense and antisense molecules wereproduced by using T7 and T3 polymerase, respectively.

Fractionations, for determining the extent of hybridization, are done byuse of the streptavidin-phenolchloroform extraction method (Sive and St.John, Nucleic Acids Res. 16:10937 (1988)). Control experiments areperformed to determine the specificity of the hybridization of thymocytesequences. Because the copy RNA (T3 products) used as driver contains˜50 nucleotides of SK(-) vector sequence, because of the position of theT3 promoter initiation site, it is important to show that thesubtractive hybridization of ssDNA is due to the presence of cloned cDNArather than vector sequences. As shown in Table 1, under the conditionsused, these vector sequences contribute only slightly to hybridization.

Since Table 1 establishes that shared cDNA sequences are effectivelyremoved, total ssDNA (see examples) is used in subtractive hybridizationto obtain cDNA for enriched library construction. Nonhybridized cDNA isconsidered to be greatly enriched for sequences present in the mRNA fromprogrammed (dying) cells but absent or rare in control cells. The ssDNArecovered after hybridization that is used in the PCR mixture consistsof SK(-) vector DNA lacking inserts, unhybridized SK(-) vectorcontaining cDNA inserts, and VCS-M13 helper phage DNA. However, only theenriched cDNA is carried forward from this mixture into the constructionof the subtracted library because the cDNA is selectively primed inconducting PCR. This amplification is performed by using the XbaI oligo(dT) primer-adaptor in conjunction with the universal M13 primer thatflanks the multiple cloning site. The effectiveness of both theamplification and subtractive hybridization is shown in FIG. 2. In thefar left lane of FIG. 2, hybridization of ssDNA with labeled cDNA fromcontrol cell mRNA is shown. The center lane of FIG. 2 (Sub) shows lackof discernible cDNA hybridization to subtracted ssDNA. One microgram ofDNA is applied in each of these two lanes. The lane at the far right ofFIG. 2 shows hybridization of cDNA to the denatured double-stranded DNAproduced by PCR upon selective priming of the ssDNA remaining aftersubtractive hybridization. Cloned cDNA that is amplified is contained inthe region of the vector as shown in FIG. 1. As the migration ofdouble-stranded DNA differs markedly from that of ssDNA, moleculardistribution in the gel is not directly comparable. Also, the PCRproduct contains concatemers. The vector and helper phage DNAs, whilepresent in the mixture, are not amplified by this procedure. ThePCR-amplified cDNA sequences, which retain their XbaI and EcoRI sites,are appropriately digested and inserted into pBluescript vector KSM13(+)to produce a library enriched for sequences that are enhanced orspecifically expressed in death-induced cells (see examples).

Screening and Selection of Clones From the Enriched, Subtracted Library

The enriched library is screened by colony blot hybridization with cDNArepresenting mRNA from control and induced (dexamethasone- andcycloheximide-treated) cells. Before use, the cDNA transcribed from mRNAfrom induced cells is subtracted by hybridization with mRNA from controlthymocytes. Using the subtracted probe, 24 hybridizing colonies areidentified from approximately 1,000 recombinants and are evaluated forduplication by cross hybridization in Southern blots.

Evaluation of Selected Clones

The evaluation of cloned cDNAs for potential correspondence to genesactivated in programmed cell death is complicated. First, the thymocytesin the control and induced cultures are not synchronous or homogeneous.In short-term control cultures, about 3 to 8% of the cells are expectedto be dying at the time we extracted RNA (Cohen and Duke, J. Immunol.132:38-42 (1984)), and therefore, are presumed to have expressed geneproducts that might be involved in the death process. In other words, atruly null control population of thymocytes is not available, even afterPercoll fractionation, and hence search and evaluation is based on mRNAsshowing either a restricted observable presence or increased abundancein induced cells. Second, following exposure to dexamethasone, all cellsin the culture do not respond synchronously (Thomas and Bell, Mol. Cell.Endocrinol. 22: 71-84 (1981)). Thus, in order to have most of the cellsentered into the death process, it is necessary to maintain them for 6to 8 h following induction. This period of time presents a problem inthat much of the mRNA in dying cells is degraded. As shown below, withinabout 4 to 6 h following induction, actin mRNA ss no longer detectibleby the standard Northern blot procedure. Therefore, to stabilize mRNAs,including those that may be the products of genes that are activated bythe inductive stimulus, cycloheximide is used to block proteinsynthesis, as previously explained. However, exposure to cycloheximidealone can result in cell death. It is found that when thymocytes aretreated with cycloheximide for 4 to 6 h and then the block is releasedby washing, increased DNA fragmentation and cell lysis are observed overthe next several hours (Sellins and Cohen, J. Immunol. 139:3199-3206(1987)). This suggests that, in immature thymus cells, cycloheximidedirectly or indirectly induces cell death processes or that theproduction of cell death messages may be constitutive at a low level;cycloheximide allows these messages to accumulate, and after the proteinsynthesis block is removed, they are expressed. Despite thesecomplications, clones of potential interest can be evaluated by usingmRNA from irradiated or dexamethasone-treated cells that are maintainedin the presence or absence of cycloheximide.

Northern Blot Analysis

Northern blots are prepared and probed with candidate cDNAs. An exampleof this further evaluation is shown for cDNAs RP-2 and RP-8 (FIG. 3). Asshown in FIG. 3, poly(A) cytoplasmic RNA (2 μg) from control (Ctl),irradiated (Irr), irradiated+cycloheximide-exposed (Irr Cyc),dexamethasone-treated (Dx), dexamethasone-cycloheximide-treated(Dx/Cyc), and cycloheximide-exposed (Cyc) cells are subjected toelectrophoresis in a 1% agarose gel. The RNA is then transferred toHybond nitrocellulose and hybridized with RP-2 cDNA. Blots are thenerased, hybridized with RP-8 CDNA, and finally erased and reprobed withactin cDNA. The sizes of hybridizing RNA species are determined bycomparison with the electrophoretic migration of RNA standards. RP-2 isfound to be complementary to a 2.1-kb mRNA and RP-8 is shown to becomplementary to a 1.3-kb mRNA present in increased abundance in bothirradiated and dexamethasone-induced cells that are maintained in thepresence of cycloheximide. Both mRNAs are also elevated in cells treatedwith cycloheximide alone. In this Northern blot, modest increasesrelative to control cell RNA are observed for RP-2 and RP-8 mRNAs fromcells treated only with dexamethasone for 4 h, but not in cells 4 hafter exposure to radiation. The effect of cycloheximide on mRNAabundance can be clearly seen by comparing the Northern blot signalsbetween stimulus-plus-cycloheximide-treated cells and cells receivingonly irradiation or dexamethasone. The lower levels of RP-2 and RP-8mRNAs in cells treated only with dexamethasone or irradiation isprobably a reflection of the mRNA abundance and integrity in general inthese populations of cells at the time RNA is harvested. This isparticularly striking in irradiated cells, where even normally abundantactin mRNA is only weakly detected.

Temporal Appearance of Candidate mRNAs

After identifying RP-2 and RP-8 as mRNAs that appear in death-inducedthymocytes, in the presence of cycloheximide, a determination is madewhether these mRNAs can be detected at various times after induction incells that are not maintained in the presence of cycloheximide. It isimportant to establish that the induction of these mRNAs is not simplyan artifact of cycloheximide treatment. Detection of these mRNAs in thecytoplasmic fraction, even without the stabilizing influence ofcycloheximide, is possible provided RNA is harvested soon afterexpression is induced, before nuclear disorganization and nucleic aciddegradation is advanced in a large number of cells in the culture.Because the time frame of early events in programmed cell death is onlyroughly known, cytoplasmic RNA is prepared from cultures at 1 hintervals following irradiation. Irradiation is elected as the inductivestimulus because it evokes a more synchronous response, and it is astimulus different from that given to cells (dexamethasone andcycloheximide) that are represented in the programmed library.

The temporal appearance of RP-2 and RP-8 mRNAs in thymocytes followinglow-dose gamma irradiation is shown in FIG. 4. Northern blot analysiswas performed on total cytoplasmic RNA isolated from thymocytes at 0, 1,2, 3, 4, 6, and 8 h after irradiation. In each lane, 20 μg of RNA isapplied. The blot is hybridized with the following cDNAs: RP-2, RP-8,actin, and an antisense 18S rRNA oligonucleotide. RP-2 mRNA appears inthymocytes 2h following irradiation but is no longer detectable after 6h. RP-8 is detectable after 1 h following exposure and likewise is nolonger detected in cells 6 h following exposure. As these cells are notstabilized by the addition of cycloheximide, actin mRNA also declinesfollowing irradiation and is only faintly detectible, if at all, after 6h. In comparison, rRNA is relatively stable throughout this period whenthe death process is ongoing. For reference, poly(A) cytoplasmic RNA(A⁺, 2 μg) in the first lane of each panel fromdexamethasone-cycloheximide-treated cells (D+C) is shown. % Fragrepresents percentages of DNA fragmentation (are determined aspreviously described (Sellins and Cohen, J. Immunol. 139:3199-3206(1985)) in the culture at various times following irradiation.

As shown in FIG. 4, the 2.1 kb RP-2 mRNA species is detectible in wholecytoplasmic RNA (mass mostly rRNA) at about 2 h following irradiation,and it is most abundant at 3 h. This is particularly striking, as at 3 hactin mRNA has already markedly declined. Thus, despite apparent highlydegradative conditions RP-2 mRNA accumulates. Using the totalcytoplasmic RNA rather than the poly(A) fraction, a variant species of˜3.4 kb is also detected in the 3-h sample. This can be another form ofthis mRNA, or it might be a primary or partially processed transcriptthat is released as a consequence of a developing nuclear lesions. At 4h, RP-2 mRNA is mostly degraded and thus appears as a smear of smallermolecules rather than a 2-kb band. By 6 h postirradiation, RP-2sequences are only faintly detectible, if at all. The presence ofhybridizing higher-molecular-weight molecules, noticeable at 4 hpostirradiation, is likely due to nuclear leakage to the cytoplasmicfraction of cleaved, genomic DNA and unprocessed primary transcripts orfragments thereof. The appearance of RP-2 mRNA seems to be late in theprogram, as it precedes or coincides with extensive breakdown ofchromatin characteristic of dying cells. As given in FIG. 4, 63% of thechromatin in the induced culture is fragmented by 4 h postirradiation.

The appearance of RP-8 mRNA is detected in advance of RP-2 when the sameNorthern blot is probed. This 1.3-kb species is elevated within 1 hfollowing irradiation. RP-8 also differs from RP-2 mRNA in that itsabundance is greatest at 1 to 2 h following induction, and it shows adecline at 3 h after induction, when RP-2 mRNA is highest. RP-8 mRNAremains detectible at 4 h, although by 4 h, just as in RP-2 sequences,hybridization reveals a smear rather than a band of molecules.

The Northern blot shown in FIG. 4 is also reprobed with actin cDNA as aninternal control for mRNA integrity. Noticeable reduction in itsabundance is detected at 3 h, and like RP-2 and RP-8 mRNAs, actin mRNAis mostly eliminated 6 h following induction of cell death. Anoligonucleotide complementary to 18S rRNA is used to probe the NorthernBlots, and as shown this relatively stable, protein-complexed RNAremained largely intact throughout the sampling period even though mRNAsare degraded.

Sequence Analysis of Death-Associated mRNAs

The initial cDNAs isolated for clones RP-8 and RP-2 are rather smallbeing 210 and 650 nucleotides in length respectively. To isolate largercDNAs and to avoid the direct sequencing of PCR-generated molecules, theprogrammed lambda libraries are screened, with RP-8 and RP-2 clones asprobes, and additional cDNA clones are obtained. For RP-8, the longestinsert obtained is 960 nucleotides and for RP-2, the longest insert isabout 1,850 nucleotides.

The nucleotide sequence and amino acid sequence of RP-8 is shown in FIG.5 (SEQ ID NO:1). There exists from the 5' end of the cDNA an openreading frame of 861 nucleotides followed by a short 3' untranslatedregion of 49 nucleotides and a poly (A) tail. The open reading framecontains a cysteine-rich region that is characteristic of zinc fingerand metallothionein proteins. A putative zinc finger of the C2H2 type(Evans and Hollenberg, Cell 52:1-3 (1988)) begins at residue 81. Thissingle zinc finger (C-XC-X12-H-X5-H) contains five amino acids betweenhistidine residues instead of the usual three found in most of thereported C2H2 zinc fingers. Proteins known to have this spacing betweenhistidine residues include hunchback (Tautz, et al., Nature 327:383-389(1987)), teashirt (Fasano, et al., Cell 64:63-79 (1991)), and the Suvar(Chirgwin, et al., Biochemistry 18:5294-5299 (1979); Compton andCidlowski, J. Biol. Chem. 262:8288-8292 (1987); Reuter, et al., Nature(London) 344:219-223 (1990)) proteins from Drosophila spp. and humanPRDII-BFI (Fan and Maniatis, Genes Dev. 4:29-42 (1990)).

A partial sequence of clone RP-2 is presented in FIG. 6 (SEQ ID NO:3).Only the portion of the 1.8-kb insert that has been sequenced in bothstrands is shown. The sequence contains an open reading frame beginningimmediately at the 5' end of the cDNA and ending 633 nucleotidesdownstream. Two additional in-frame stop codons are also found 6 and 12nucleotides from the first terminations signal. The 3' untranslatedregion of RP-2 is quite long, being about 1,200 nucleotides in length,of which only 218 nucleotides is shown in FIG. 6 (SEQ ID NO:3). The cDNAclone also contains a poly(A) tail and polyadenylation signal. RP-2 cDNAis expressed in a bacterial expression vector, and the size of theinduced fusion protein is consistent with the length of the open readingframe predicted from cDNA sequencing. Structural analysis of the openreading frame classifies RP-2 as an integral membrane protein (Klein, etal., Biochim. Biophys. Acta 815:468-476 (1985)) with a 17 amino-acidhydrophobic membrane-spanning domain and a 41-amino acid cytoplasmicdomain. A membrane-associated alpha helix predicted by several methods(Eisenberg, et al., J. Mol. Biol. 179:125-142; Rao and Argos, Biochim.Biophys. Acta 869:197-214 (1986)) runs from glycine 152 to proline 170.Two potential N-glycosylation sites are located upstream from themembrane-spanning domain.

In Situ Hybridization of RP-8

A. Probe Labeling

Synthetic oligonucleotide anti-sense probes for RP-8 (20-22 mers) areused for in situ hybridization experiments. Probes are labeled at the3'-end with ³⁵ S-dATP (Amersham) with terminal transferase (Strategene).After the labeling reaction, labeled oligonucleotides are separated fromunincorporated ³⁵ S-dATP by purification on a Sephadex G-25 "spincolumn" (Boehringer Mannheim). This method is used to label probes withspecific activities between 2-5×10 dpm/ug. For control hybridizations,oligonucleotide sense probes are labeled. The control probes allowassessment of the extent of non-specific binding.

B. Preparation Of Slides, Hybridization, And Washing

Epithelial sections are floated off the slides in 1X SSC. The sectionsare then placed in 25-50 ul of a hybridization solution consisting of 4XSSC with 50% deionized formamide, 1x Denhardt's solution with 10%dextran sulfate, sonicated salmon sperm DNA (500 ug/ml), 10 mMdithiothreitol, and 250,000 cpm/100 ul of labeled oligonucleotide for12-16 hours at 32° C. The sections are transferred to petri dishes andwashed for 30 minutes in 1X SSC at room temperature, next, in 0.2X SSCat 38° C., and finally in 0.2 X SSC at room temperature. Sections aremounted on slides for autoradiography.

Expression of RP-8 In Cells

Using nucleotide sequences contained within the RP-8 protein encodingregion as probes in an in situ hybridization procedure, expression isobserved of the RP-8 protein in several types of cells discussed below.

The RP-8 sequence is used to probe granular neurons in the cerebellumthat undergo cell death in the mutant mouse (weaver). Cell death occursduring early postnatal development in these animals (Symyne andGoldwitz, J. Neurosci. 9:1608-1620 (1989)). The RP-8 sequence is foundby in situ hybridization, to be expressed in cells positioned in theexternal granular layer of the cerebellum.

In the normal adult liver a low level of programmed cell death (asassumed from the presence of apoptotic cells) occurs. RP-8 expression isobserved in fewer than one cell in about 5000. The rare expression ofRP-8 is consistent with the frequency of the presence of apoptoticcells.

The thymus gland from adult rats that are untreated or treated withglucocorticoid are examined by in situ hybridization. Glucocorticoidsraise the level of PCD in the thymic lymphocytes. Expression of RP-8 isobserved to be increased about 2-fold in thymus glands from the steroidtreated animals. Thus, increased expression (number of cells with RP-8sequences) is linked with increased programmed cell death in the thymusgland.

Sensory cells of the olfactory region of the nasal passageway arecontinuously renewed. Some of the new sensory cells fail to migrate andestablish functional connections with olfactory nerve fibers and theydie. When the olfactory epithelium is probed, expression of RP-8 isobserved in cells located in the region where programmed cell death isthought to occur in cells that fail to migrate.

These examples indicate that RP-8 expression is a marker for the processof programmed cell death in several types of cells in addition tothymocytes.

Deposit Of Strains Useful In Practicing The Invention

A deposit of biologically pure cultures of the following strains wasmade with the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md., the accession number indicated was assigned aftersuccessful viability testing, and the requisite fees were paid. Accessto said culture will be available during pendency of the patentapplication to one determined by the Commissioner to be entitled theretounder 37 C.F.R. Section 1.14 and 35 U.S.C. Section 122. All restrictionon availability of said culture to the public will be irrevocablyremoved upon the granting of a patent based upon the application andsaid culture will remain permanently available for a term of at leastfive years after the most recent request for the furnishing of a sampleand in any case for a period of at least 30 years after the date of thedeposit. Should the culture become nonviable or be inadvertentlydestroyed, it will be replaced with a viable culture(s) of the sametaxonomic description.

The RP-8 cDNA sequence, inserted into the plasmid pBluescript SKM13(-)in the strain E. coli XL-Blue, has been given ATCC No. 69031 and depositdate, Jul. 14, 1992. The RP-2 cDNA sequence, inserted into the plasmidpBluescript SKM13(-) in the strain E. coli XL-Blue, has been given ATCCNo. 69032 and deposit date, Jul. 14, 1992.

The RP-2 and RP-8 genes and their gene products, as will be apparent tothose of skill in the art, are useful in the treatment and diagnosis ofhuman diseases. In the treatment of a particular disease, such ascancer, they are useful in that the selective activation of the genesleads to the expression of proteins which signal the onset of theprogrammed cell death for the cancer cells. Another use for the RP-2 andRP-8 genes is their selective elimination of immune cells linked toautoimmune diseases. As in the cancer cells, selective activation of theRP-2 and RP-8 genes results in expression of a protein that eliminatesthose immune cells active in the autoimmune disease. A therapy whichutilizes the body's own genes such as RP-2 and RP-8 to selectively treatcancer can lessen or eliminate the need for chemotherapy. Chemotherapyis not selective and often weakens the patient more than the cancer.

A knowledge of the RP-2 and RP-8 gene products are useful in designingdrugs for treatment of neurodegenerative diseases. Drugs designed toinhibit the activity of these gene products can block unwanted celldeath a it occurs in neurodegenerative diseases. In the case of anantibody designed drug there is inhibition of the protein activity. Withan oligonucleotide designed drug, an ologonucleotide is designed to becomplementary to the mRNA of the gene associated with the unwanted celldeath. Inhibition of mRNA activity occurs by hybridization-basedblockage of translation or the degradation of specific mRNAs.

RP-2 and RP-8 and their gene products are of considerable use inmonitoring the extent of cell death associated with specific diseases.Once the severity of the disease is assessed from the amount of RP-2 orRP-8 gene product present, a physician can then determine the course oftreatment.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

EXAMPLE 1 Preparation And Culture Of Thymocytes

Single-cell suspensions were prepared and cultured from the thymuses of4- to 6-week-old male Sprague-Dawley rats by using the procedures andtissue culture medium previously described (Sellins and Cohen, J.Immunol. 139:3199-3206 (1987)). Steroid-induced thymocytes were culturedfor the indicated periods in the presence of 10⁻⁶ M dexamethasone (Cohenand Duke, J. Immunol. 132:38-42 (1984)). Where indicated, 50 μg ofcycloheximide per ml was also included in the culture medium. Thymocytesexposed to irradiation received 900 R from a ⁶⁰ Co source (Sellins andCohen, J. Immunol. 139:3199-3206 (1987)).

EXAMPLE 2 Isolation Of Total Cytoplasmic RNA From Thymocytes

Total cytoplasmic RNA was isolated from thymocytes by using the protocolof either Chirgwin et al. (Biochemistry 18:5294-5299 (1979)) orChomczynski and Sacchi (Anal. Biochem. 162:156-159 (1987)), followingthe removal of nuclei by low-speed centrifugation. Polyadenylated RNAwas obtained by oligo(dT) chromatography, and total cellularpolyadenylated RNA was obtained by using the Fast Track mRNA kit(Invitrogen, San Diego, Calif.). RNA was prepared from controlthymocytes immediately after the preparation of single-cell suspensions.

EXAMPLE 3 Construction Of Directionally Cloned cDNA Libraries FromThymocytes

Directionally cloned cDNA libraries were constructed by usingpolyadenylated cytoplasmic RNA from control thymocytes and thymocytescultured 8 h in the presence of dexamethasone and cycloheximide. Tominimize programmed cell death in the control population animals wereadrenalectomized 2 weeks prior to RNA isolation. Also, at the time ofharvest, apoptotic cells were removed from the control cell suspensionsby Percoll gradient sedimentation (Wyllie and Morris, Am. J. Pathol.109:78-87 (1982)).

First-strand cDNA was synthesized by using an oligo(dT) primer-adapter(Promega, Madison. Wis.) containing an XbaI restriction site 5' to theoligo(dT) tail. Reaction mixtures (200 μl) contained 5 to 10 μg ofpolyadenylated RNA in standard reverse transcriptase buffer (50mMTris-HCl, pH 8.0, 8 mM MgCl₂, 50mM KCl, 1 mM dithiothreitol, 50 μg ofRNase-free bovine serum albumin per ml), 5 mM deoxynucleosidetriphosphate (dNTP), 12.5 μg of primer per ml, 1 U of human placentalRNase inhibitor per μl, 50 μg of actinomycin D per ml, and 20 to 25 U ofreverse transcriptase (Life Sciences Inc.). To monitor synthesis, 20 μCiof [³² P]dCTP (3,000 Ci/mmol) was included in the reaction mixture.Second-strand cDNA was synthesized by using the procedures described byGubler and Hoffman (Gene 25:263-269 (1983)).

The cDNA was blunt ended by using T4 polymerase (Bethesda ResearchLaboratories) and then methylated by using EcoRI methylase (New EnglandBioLabs). EcoRI linkers radioactively labeled and phosphorylated withpolynucleotide kinase (Boehringer Mannheim) were ligated to 200 to 500ng of cDNA with T4 DNA ligase (New England BioLabs) during a 12 hincubation at 15° C. Following the ligation of linkers, reactions werediluted and digested sequentially with EcoRI and XbaI. Digested linkerswere separated from cDNA by three ethanol precipitations in the presenceof 2.2 M ammonium acetate. pH 5.2, followed by one precipitation in 0.3M sodium acetate, pH 5.2. Digestion of linkers was monitored byelectrophoresis in 2% agarose gels and autoradiography. A second roundof digestions was often required to completely cut the ligated linkers.Final cDNA pellets were resuspended in 10 to 20 μl of sterile 0.1×TEbuffer (1×TE buffer is 10 mM Tris-HCl, pH 7.4,2 mM EDTA).

Different amounts of double-stranded cDNA were ligated to appropriatelydigested Lambda Zap I (Stratagene Cloning Systems, La Jolla, Calif.) byusing their recommended conditions. Ligated vector DNA was then insertedinto phage particles by using the Gigapak Plus-6 packaging system(Stratagene Cloning Systems). Amplified stocks were prepared frominfected Escherichia coli BB4 (Maniatis, et al., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1982)).

A second cDNA library from programmed cells was also prepared as justdescribed except that NotI linkers were used to clone cDNA into LambdaZap. This library was used to isolate larger cDNA inserts.

EXAMPLE 4 Preparation Of Phage DNA

Synthesis and isolation of phagemid DNA was essentially done byfollowing the Stratagene protocol. The host E. coli strain XL1-Blue wasgrown in 2×YT medium supplemented with tetracycline (12.5 μg ml). At anA₆₀₀ of 0.4 to 0.5 (3×10⁸) to 5×10⁸ bacteria per ml), exponentiallygrowing cells were coinfected with a 10-fold excess of lambda phageparticles from the programmed library and a 5-fold excess of VCS-M13helper phage. After a 30-min incubation, infected bacteria were dilutedinto 1 liter of fresh prewarmed medium supplemented with tetracycline.To select for bacteria infected with both viral particles, theantibiotics kanamycin (25 μg/ml) and ampicillin (100 ug/ml) were addedto the cultures 30 min later. Cultures were then incubated an additional8 to 16 h before phage particles were harvested.

To isolate phage DNA, bacterial cells were pelleted and phage-containingsupernatants were precipitated with one-fourth volume of 3.5 M ammoniumacetate, pH 7.5, containing 20% polyethylene glycol at 4° C. To ensurethat all the expected DNA molecules were synthesized, DNA extracted froman aliquot of the precipitated material was analyzed on agarose gelsbefore the phagemid and lambda phage particles were separated by CsClgradient centrifugation.

The precipitated phage were collected by centrifugation for 15 min at10,000×g and resuspended in 20 ml of SM buffer (50 mM Tris-HCl, pH 7.5,20 mM MgSO4, 100 mM NaCl, 0.1% gelatin). Solid CsCl was added to 0.5g/ml, and phage particles were banded by centrifugation at 4° C. for 18to 24 h in a Beckman SW41 rotor at 30,000 rpm. Phagemids collected as aviscous band near the top of the gradient, whereas lambda particles werefound in a lower more diffuse band. Phagemids were dialyzed againstbuffer (50 mM Tris-HCl, pH 8.0, 10 mM NaCl; 10 mM MgCl₂) for 2 to 3 h atroom temperature. The dialyzed material was then treated for 15 min at37° C. with 2 μg of pancreatic DNase and RNase per ml, followed by theaddition of EDTA (20 mM, pH 8.0). 0.5% sodium dodecyl sulfate (SDS), and50 μg of proteinase K per ml. After 30 min at 65° C., extractions withphenolchloroform and chloroform were performed and the DNA was collectedby ethanol precipitation. From 1 liter of culture up to 1 mg of closedcircular single-stranded DNA (ssDNA) was obtained. The ssDNA consists ofVCS-M13+ helper phage DNA, ssDNA containing cloned cDNA sequences, and asmall amount of SK(-) ssDNA lacking inserts. ³ H-labeled ssDNA wasprepared from bacterial cells grown in the presence of [methyl-³H]thymidine (1 μCi/ml; specific activity=6.7 Ci/mmol). ¹²⁵ I-labeledssDNA was prepared by using the protocol of Chikaraishi et al. (Cell13:111-120 (1978)).

Closed circular ssDNA lacking cDNA inserts [SK(31 )ssDNA] was obtainedby infecting XL1-Blue cells transformed with the pBluescript plasmidSK(-) (Stratagene Cloning Systems) with VCS-M13 helper phage. After anovernight incubation, phagemid DNA was purified by using the Stratageneprotocol for isolation of ssDNA. Helper phage DNA was separated fromSK(-) ssDNA by chromatography on Sephacryl S-1000 (see FIG. 3, lane c).VCS-M13 ssDNA was prepared from host XL1-Blue cells infected with helperphage alone and was purified as just described. The large-scalepreparation of lambda DNA from the amplified control library phage stockwas done by using standard procedures (Maniatis, et al., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1982)).

EXAMPLE 5 In Vitro RNA Transcription

Purified lambda DNA synthesized from the control library was linearizedwith EcoRI when T3 polymerase was used and XbaI when T7 polymerase wasused. Digestions were repeated twice to ensure complete linearization oftemplate. After the second digestion, reactions were incubated 15 min at37° C. with proteinase K (50 μg/ml), followed by extraction withphenolchloroform and chloroform alone. Digested template DNA wascollected by ethanol precipitation and resuspended in diethylpyrocarbonate (DEPC)-treated water at 0.5 to 1 μg/ml.

Transcription reactions (200 μl) were routinely done with 15 to 20 μg oftemplate DNA in commercially provided transcription buffer containing1.125 mM NTPs, RNase inhibitor (1 U/μl), and 1 mM dithiothreitol. T3polymerase (Stratagene Cloning Systems} or T7 polymerase (BethesdaResearch Laboratories) was added (50 U per reaction), and mixtures wereincubated 90 min at 37° C. An additional 100 U of RNase inhibitor and 50U of polymerase were added, and incubations were continued an additional60 min. Samples were then treated with 20 U of RNAse-free DNase for 30min, and the synthesized RNA was purified by phenolchloroform extractionand ethanol precipitation. Precipitated RNA was quantitated by A₂₆₀ andstored at -70° C. in DEPC-treated water. From a typical reactioncontaining 20 ,ug of template DNA, up to 100 μg of copy RNA wasobtained. RNA (50 to 100 μg) was labeled with photobiotin (SigmaChemical Co.) according to the protocol of Welcher et al. (Nucleic AcidsRes. 14:10027-10043 (1986)) except that the amount of photobiotin usedin the reaction was reduced to half the mass of the RNA.

EXAMPLE 6 Subtractive Hybridization

To construct the subtracted library, biotinylated copy RNA (150 μg) andssDNA preparation (30 μg), of which one-fourth was estimated to be ssDNAcontaining cDNA inserts were coprecipitated in ethanol with 0.3 M sodiumacetate, pH 5.2. Precipitates were collected in siliconizedmicrocentrifuge tubes (14,000×g for 15 min), washed with 70% ethanol inTE buffer, and repelleted. Final pellets were dried and dissolved in2×PIPES hybridization buffer {0.1 M PIPES[piperazine-N,N'-bis(2-ethanesulfonic acid)], pH 6.8, 1.2 M NaCl, 2 mMEDTA, 0.2% SDS}, and an equal volume of deionized formamide was added tothe hybridization mixture. Samples were heated at 95° C. for 1 min andthen incubated at 42° C. until the desired C₀ t value was achieved.Alternatively, material was first dissolved in H₂ O and then dilutedwith an equal volume of 2×hybridization solution. Hybridizations inthese solutions were performed at 65° C. in sealed glass capillarytubes.

To separate unhybridized (subtracted ssDNA) from hybridized sequences,reactions were diluted 10 to 15 times with streptavidin binding buffer(10mM Tris-HCl, pH 7.4, 400mM NaCl, 2 mM EDTA) and incubated withstreptavidin (Sigma Chemical Co.) for 30 min at 60° C. Streptavidin wasadded at a mass equal to the mass of copy RNA driving the hybridization.An equal volume of equilibrated phenolchloroform was added to themixture, and phases were separated by centrifugation. The aqueous phasewas carefully withdrawn and subjected to a second phenol-chloroformextraction followed by a final chloroform extraction. When thesubtracted ssDNA was used for polymerase chain reaction (PCR)amplification and cloning, the subtracted material was either treatedwith RNase A for 15 min at 37° C. or incubated 60 min at 65° C. in 0.2 NNaOH to remove trace amounts of copy RNA. Material was againphenol-chloroform and chloroform extracted and recovered by ethanolprecipitation with yeast tRNA added as a carrier. Final pellets wereresuspended in 10 to 20 μ l of 0.1×TE buffer and stored at -20° C.

EXAMPLE 7 PCR Amplification Of Subtracted ssDNA

Approximately 2 to 5 ng of subtracted ssDNA from the programmed librarywas amplified by PCR (Saiki, et al., Science 230:1350-1354 (1985)).Reaction mixtures contained Taq polymerase buffer, 0.2mM each dNTPs, 25to 30 pmol of primers, and 2.5 U of Taq polymerase. Primers used for thespecific amplification of cloned sequences were the oligo(dT)-XbaIprimer used in first-strand cDNA synthesis and the universal M13sequencing primer. Primer annealings were conducted at 31° C. for 5cycles followed by 25 cycles at 48° C. Amplified PCR-generated DNA waspurified by extraction first with phenol-chloroform and then withchloroform.

EXAMPLE 8 Construction And Screening Of Subtracted Libraries

The PCR-amplified sequences were digested with both XbaI and EcoRI andligated into similarly digested pBluescript vector KSM13(+). Ligationreactions were diluted fourfold with sterile H20, and small amounts wereused to transform high-efficiency XL1-Blue competent cells (StratageneCloning Systems) or DH5α competent cells (Bethesda ResearchLaboratories) by using the supplier's recommended protocol. To screenthe subtracted library, transformation mixtures were plated directlyonto nitrocellulose filters overlaid onto agar plates containing theappropriate antibiotics, 10 mM IPTG(isopropyl-β-D-thiogalactopyranoside) and 80 U of X-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) per ml. Replicatefilters were prepared by direct transfer of bacterial colonies onto asecond prewetted nitrocellulose filter (Maniatis, et al., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1982)). A master plate andtwo filters were prepared in this manner from each transformation. Cellswere prepared for hybridization by the protocol of Grunstein and Wallis(Methods Enzymol. 68:379-389 (1979)). In some screens, individualcolonies of different transformations were consolidated onto a singleplate from which replicate filters were prepared for use inhybridization with probe cDNA. Filters were first incubated for 4 to 6 hat 42° C. in hybridization solution containing 50% formamide, 4×SSPE(1×SSPE is 0.18 M NaCl, 10 mM NaPO4) and 1 mM EDTA, pH 7.7), buffer,0.1% SDS, herring sperm DNA (200 μg/ml) and 50x Denhardt's solution, andthen hybridization was conducted for 48 to 72 h (6 ml/150 mm filter) insolution containing 1×10⁶ to 2×10⁶ cpm of control cDNA (specificactivity=3×10⁸ to 5×10⁸ dpm/μg) per ml or 0.5×10⁶ to 1×10⁶ cpm ofsubtracted cDNA (5×10⁸ to 8×10⁸ dpm/μg) per ml. RNA isolated fromthymocytes incubated for 8 h with both dexamethasone and cycloheximidewas used in preparing the subtracted cDNA probe. Followinghybridization, filters were washed at increasing temperatures in2×SSC-0.02% SDS and 0.2×SSC-0.2% SDS (1×SSC is 0.15 M NaCl plus 0.015 Msodium citrate). Final stringency washes were done for 30 min at 65° C.

EXAMPLE 9 Synthesis Of cDNA Probes

To synthesize cDNA for probing the subtracted library. 1 to 2 μg ofpolyadenylated RNA was collected by ethanol precipitation andresuspended in 5 μl of DEPC-treated water containing 0.4 ,ug ofoligo(dT) primer. After being heated 5 min at 65° C., RNA and primerwere transferred to a tube containing 200 to 400 μCi of [³² P]dCTP(>3,000 Ci/mmol). 40 μM cold dCTP, and 0.5 mM remaining dNTPs, 2 U ofRNase inhibitor per μl, reverse transcriptase buffer,. 50 μg ofactinomycin D per ml. and 5 U of reverse transcriptase. The finalreaction volume was 20 μl, and reactions were incubated 60 min at 37° C.Probes were purified by phenol-chloroform extractions and ethanolprecipitation in the presence of 2.2 M ammonium acetate. The cDNA wasthen incubated 1 to 2 h in 0.1 N NaOH at 65° C., neutralized, andreprecipitated in ethanol. Probes synthesized from control ceil RNA wereresuspended in a small volume of water and added directly to thehybridization solution. Subtracted probes were prepared by hybridizingcDNA generated from mRNA of cells incubated for 8 h in media containingdexamethasone and cycloheximide, the experimental cell mRNA, to a10-fold excess of control thymocyte mRNA until a C₀ t value of 1,200 to1,700 was attained. Unhybridized cDNA was recovered by hydroxylapatitechromatography. alkali treated, and precipitated by the addition ofethanol.

EXAMPLE 10 Northern (RNA) Blot Analysis

Indicated amounts (see figure legends) of total cytoplasmic orpolyadenylated cytoplasmic RNA were separated in 1.0 to 1.2% agarosegels and transferred to nylon membranes as described by Fourney et al.(Focus 10:5-7 (1988)).

EXAMPLE 11 DNA Sequence Determination

cDNA clones were sequenced by the dideoxy-chain termination protocolusing a modified form of T7 polymerase (Sequenase; U.S. Biochemical).Primary cDNA sequences were derived from clones isolated from the"death-induced" Lambda Zap libraries, not the PCR-generated subtractedlibraries, and sequences presented here were obtained by sequencing bothcDNA strands. This was achieved by sequencing overlapping subclones fromthe plasmid pKSM13(+) and by using cDNA-specified oligonucleotides asprimers.

All sequence analyses and identification of structural motifs were donewith the PC/Gene software program (Intelligenetics, Inc.). The mostupdated GenBank and EMBL nucleic acid data banks and the Swiss-Protprotein data bank were searched through the Internet network by usingthe FASTA program of Pearson and Lippman (Proc. Natl. Acad. Sci. USA85:2444-2448 (1988)).

EXAMPLE 12 Expression Of RP-8 In Thymus

The expression of RP-8 genes in vivo before and after treatment withdexamethasone was examined to determine if RP-8 was present in allthymocytes or restricted to a subset of the population. Therefore, insitu hybridization was used to follow both the temporal and spatialdistribution of these mRNAs.

Rats or mice were injected intraperitoneally with 5 mg dexamethasone perkg body weight and were kept up to 270 minutes before being sacrificedby CO₂. Thymuses were rapidly dissected and fixed by immersion inbuffered 4% paraformaldehyde. Tissue was frozen and sectioned forhybridization with sense and antisense oligonucleotides. The antisenseoligos represent RP-8 sequences that are 95-100% homologous between ratand mouse. When thymic sections from a control and experimental mousewere hybridized to a RP-8 antisense oligonucleotide, a hybridizationpattern was shown. Clusters of RP-8 positive cells were observed incortical regions of the thymus, particularly at the border of cortex andmedulla. Occasionally, labeled cells were also found well within themedulla. There was a dramatic increase in the number of RP-8 labeledcells at 120 minutes after dexamethasone injection. In a morequantitative study, similar but less dramatic increases in RP-8 mRNAwere observed in rat thymuses from about 90-270 minutes after steroidinjection.

In both the mouse and rat, RP-8 was detected in isolated single cellsand also in groups of adjacent cells. By looking at sections lessexposed than those shown, there appeared to be 3-4 cells per group.Single labeled cells and cell clusters were scattered amongst mostlyunlabeled cells. Several controls were used to establish the specificityof hybridization to the RP-8 antisense oligonucleotide. Thehybridization signal in the thymus was competed out with cold antisenseRP-8 oligonucleotide and a sense oligonucleotide of similar Tm did notdisplay this pattern of hybridization. Additionally, a similarhybridization pattern was observed using an antisense oligo representinga different portion of the RP-8 sequence.

The intense pattern of hybridization seen in the thymus was not observedin a normal adult liver, a tissue that under normal conditions turnsover at a very slow rate. Typically, 0-2 labeled cells per section wereseen (about 5,000 cells) which is similar to the number of apoptoiccells (0-2 per 2,000-3,000 cells) reported for normal liver (Bursch etal., Carcinogenesis 11:847-853 (1990)). Based on their location, most ofthe labeled cells appeared to be hepatocytes although the possibilitycannot be eliminated that at least some of the labeled cells in theliver might be infiltrating lymphocytes. With the control sense probe,no labeled cells were detected in three different sections (15,000cells).

RP-8 expression in different adult tissues also has been observed byNorthern blot analysis. RP-8 was detected in every tissue examined(brain, liver, kidney, lung, muscle, heart, and spleen) at levels belowthat found in thymocytes.

The pattern of RP-8 expression in thymus is consistent with theappearance of apoptosis. The mRNA is not present in most corticalthymoctes, but rather is found in isolated cells or in small groups ofcells. In vivo administration of steroids results in an increase in RP-8cells that is similar to the twofold induction of RP-8 mRNA observed invitro. Most RP-8 expressing cells in untreated thymocytes were found atthe interface between cortex and medulla. The majority of immaturethymocytes within the thymus populate the cortex whereas mature T cellsare found in the medulla. The junction between cortex and medulla mayrepresent a zone of normal cell death with the thymus. When observed,RP-8 levels appear very high within individual cells expressing themRNA. In tissues such as liver and adult brain where cell death normallyoccurs infrequently, only a few cells expressing RP-8 mRNA weredetected.

EXAMPLE 13 RP-8 Expression in Weaver Mice Correlated With Death OfGranule Cell Precursor Cells

An example of cell death in the CNS is the death of precursors togranule cell neurons that occur in mice homozygous for the weavermutation. Progenitors to granule cell neurons proliferate normally inthe external germinal layer (EGL) of weaver mice but fail todifferentiate and migrate to their normal position in the internalgranule cell layer. Consequently, the majority of these cells die byapoptosis in the inner region of the EGL (Smyne & Goldwitz, J. Neurosci.9:1608-1620 (1989)) or just below it in the molecular layer. Deathbegins during the first postnatal week and continues until about P20.

When cerebellums from 8-10 day old weaver mice were probed with the RP-8antisense probe, heavily labeled cells were scattered throughout theinner regions of the EGL. Consistent with the cessation of cellproliferation and death in weaver cerebellum, RP-8 positive cells werenot as numerous by P17 and absent by P24. Labeled neurons were alsofound in the globus pallidus, striatum, and substantia nigra of 10 dayold weaver mice.

These results clearly correlate RP-8 expression with cell death in theCNS. The appearance of RP-8 positive cells is precisely wheredegenerating granule cell progenitors are seen in weaver cerebellum. Theidentity of RP-8 positive cells as granule cells is confirmed by doublelabeling experiments using an antibody to the cell surface glycoproteinL1. Of further significance is the expression of RP-8 in isolatedneurons of the substantia nigra and striatum, a second region of knowncell death in weaver mice (Graybiel and Roffler-Tarlov, J. Neurosci.10:720-733 (1990)). Histological stains confirm the presence ofapoptotic cells in the globus pallidus since extensive cell death hasnot be reported in this region.

EXAMPLE 14 RP-8 Expression In the Rat Olfactory Epithelium

There is a continuous turnover of cells in the adult olfactoryepithelium (Graziadei & Monte-Graziadei, J. Neurocytology 8:1-18(1979)), particularly in the basal region where newly generated immatureolfactory receptors are located (Farbman, et al., J. Neuroscience.8:3281 (1988)). To determine whether RP-8 was expressed in a subset ofolfactory cells, tissue sections also were probed through the ratolfactory epithelium.

Labeled cells were observed in cells near the basal layer of theolfactory epithelium. Labeled cells were not seen in this region with acontrol sense probe.

EXAMPLE 15 RP-8 Expression In Hematopoietic Cell Lines

The cell lines surveyed for RP-8 expression included S49.1 cells andCTLL-2s. S49.1 cells are a murine lymphoma cell line that are killed byglucocorticoids (Wyllie et al, Int. Rev. Cytol. 17:755-785 (1984)) andCTLL-2s are a killer T cell line dependent on IL-2 for proliferation andsurvival (Duke and Cohen, J. Immunol. 132:38-42 (1986)).

Small increases in RP-8 mRNA were observed 10-16 hours after inductionin both cell lines. RP-8 mRNA was observed in untreated cells from bothlines. In situ hybridizations determine that RP-8 expression inuntreated cells is restricted to a small fraction of the total cellpopulation. This result is consistent with the low level of deathongoing in these cell lines.

EXAMPLE 16 Synthesis Of RP-8 and RP-2 Fusion Proteins Using BacterialExpression Vectors

Several bacterial expression vectors were available in which the RP-8and RP-2 cDNAs were expressed. The vectors that were used to generateRP-8 fusion proteins for antibody preparation are summarized below.

pET Expression Vectors

In these plasmid expression vectors cDNA sequences are cloned behind aT7 phage promoter and initiation codon and are thus transcribed by T7RNA polymerase, a highly efficient and specific RNA polymerase(Rosenberg et al, Gene. 56:125 (1987)). Large quantities of fusionprotein are obtained using this expression system. The specific vectorfor expression, peT5t, has multiple cloning sites. Fusion proteinssynthesized from this vector contain 12-28 amino acids of vectorsequence and are highly insoluble. Milligram quantities of severalfusion proteins have been purified from inclusion bodies (Nagai andThogerson, Methods in Enzym. 153:461 (1987)) derived from thisexpression system.

pBluescript Vectors

These vectors, commercially available from Stratgene Inc. are alsoexpression vectors in which transcription of cDNAs are under control ofthe lac promoter. Fusion proteins are induced by incubation for 3-4hours with IPTG. This vector has been used to obtain RP-8 fusion proteinfrom inclusion body preparations.

RP-8 fusion protein has already been purified in mg quantities fromstandard inclusion body preparations and solubilized in detergent buffer(Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, N.Y. (1988)). If additional purification is required fromthat described above, elution from polyacrylamide gels or conventionalchromatography is employed. Methods for immunization and antibodypurification are well established (Harlow & Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1988)).Immunizations are done at a minimum of 4 week intervals using 100-200 ugpurified fusion protein suspended in RIBI adjuvant (RIBI Research Inc.,Hamilton, Mont.). Injections and bleedings are taken 10-14 days afterimmunizations. To obtain the cleanest possible reagents for EM and lightmicroscopy studies, affinity purified antibodies are obtained fromantisera raised in rabbits using purified fusion protein as theabsorbent by coupling of the antigen to an activated support such asSepharose CL-4B. Purified fusion proteins are used to generatemonoclonal antibodies to obtain highly specific RP-8 antibodies.

To obtain antibodies to RP-2 and RP-8, the cDNAs were cloned in thecorrect translational reading frame into bacterial expression vectors.RP-8 was cloned into the pBluescript SKM13(-) vector from Stratagene,Inc. and RP-2 was cloned into both pBluescript SKM13(-) and a derivativeof the pPET expression vectors described by Studier. The RP-8 constructencoded an IPTG inducible fusion protein of 40 kd and RP-2 encoded a 26kd fusion protein. Taking into account that 25 amino acids of the hybridprotein represent B-galactosidase, sequences the size of the RP-8 fusionprotein are close to that predicted from the calculated molecular weight(32,000) of the RP-8 cDNA open reading frame. Similarly theelectrophoretic migration of the RP-2 fusion protein is what would beexpected, based on the calculated molecular weight of 23 kd for RP-2.Milligram quantities of fusion protein were then purified from inclusionbody preparations and injected intramuscularly into rabbits using RIBIadjuvant. The immune sera for each protein is used to characterize andpurify the relevant proteins from the thymus.

As will be apparent to those skilled in the art in which the inventionis addressed, the present invention may be embodied in forms other thanthose specifically disclosed above without departing from the spirit oressential characteristics of the invention. The particular embodimentsof the present invention described above, are, therefore, to beconsidered in all respects as illustrative and not restrictive. Thescope of the present invention is as set forth in the appended claimsrather than being limited to the examples contained in the foregoingdescription.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 972 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                            (B) LOCATION: 1..864                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCGCTCGCCTTCCTGCTGCAAGTGTACGCACCGCTTCCGGGTCGGGAC48                            ProLeuAlaPheLeuLeuGlnValTyrAlaProLeuProGlyArgAsp                              1 51015                                                                       GACGCCTTCCACCGTAGCCTCTTTCTCTTCTGCTGTCGAGAGCCGCTG96                            AspAlaPheHisArgSerLeuPheLeuPheCysCysArgGluProLeu                               202530                                                                       TGTTGCGCCGGCCTGCGAGTTTTTCGTAATCAGCTACCAAGGAAAAAT144                           CysCysAlaGlyLeuArgValPheArgAsnGlnLeuProArgLysAsn                               354045                                                                       GCATTTTACTCATATGAGCCCCCTTCTGAAACGGGAGCTTCGGATACA192                           AlaPheTyrSerTyrGluProProSerGluThrGlyAlaSerAspThr                              5 05560                                                                       GAATGTGTGTGCCTCCAACTTAAGTCTGGAGCTCATCTCTGCAGGGTT240                           GluCysValCysLeuGlnLeuLysSerGlyAlaHisLeuCysArgVal                              65 707580                                                                     TGTGGTTGCTTGGCCCCTATGACATGCTCTAGGTGCAAACAGGCACAT288                           CysGlyCysLeuAlaProMetThrCysSerArgCysLysGlnAlaHis                               859095                                                                       TACTGCAGCAAGGAACATCAGACATTAGACTGGCAGCTCGGCCACAAG336                           TyrCysSerLysGluHisGlnThrLeuAspTrpGlnLeuGlyHisLys                               100105110                                                                    CAGGCTTGTACACAGTCAGACCATTTAGACCATATGGTTCCAGACCAT384                           GlnAlaCysThrGlnSerAspHisLeuAspHisMetValProAspHis                               115120125                                                                    AACTTGCTGTTTCCAGAATTTGAAATTGTAACAGAAACAGAAGACGAG432                           AsnLeuLeuPheProGluPheGluIleValThrGluThrGluAspGlu                              13 0135140                                                                    ATTGGGCCTGAGGTGGTGGAAATGGAGGATTACTCTGAAGTTATAGGA480                           IleGlyProGluValValGluMetGluAspTyrSerGluValIleGly                              145 150155160                                                                 AGCATGGAGGGAGTACCTGAGGAAGAACTAGATTCCATGGCAAAGCAT528                           SerMetGluGlyValProGluGluGluLeuAspSerMetAlaLysHis                               165170175                                                                    GAATCCAAGGAAGATCACATATTCCAAAAGTTTAAATCCAAAATAGCC576                           GluSerLysGluAspHisIlePheGlnLysPheLysSerLysIleAla                               180185190                                                                    CTTGAACCAGAGCAGATTCTCAGGTATGGAAGAGGTATTAAACCCATC624                           LeuGluProGluGlnIleLeuArgTyrGlyArgGlyIleLysProIle                               195200205                                                                    TGGATTTCTGGTGAAAATATTCCTCAAGAAAAAGATATTCCAGATTGC672                           TrpIleSerGlyGluAsnIleProGlnGluLysAspIleProAspCys                              21 0215220                                                                    TCATGTGGTGTTAAGAGAATATTTGAATTCCAGGTCATGCCTCAGCTG720                           SerCysGlyValLysArgIlePheGluPheGlnValMetProGlnLeu                              225 230235240                                                                 TTGAACCACCTTAAGGCAGACAGACTGGGTACAAGTGTGGACTGGGGC768                           LeuAsnHisLeuLysAlaAspArgLeuGlyThrSerValAspTrpGly                               245250255                                                                    ATCTTGGCTGTCTTCACCTGTGCTGAGAGCTGCAGCCTGGGCATCGGG816                           IleLeuAlaValPheThrCysAlaGluSerCysSerLeuGlyIleGly                               260265270                                                                    TTCACAGAAGAATTTGTGTGGAAACAGGATGTGACAGAGACACCATGAAAGGTG871                     PheThrGluGluPheValTrpLysGlnAspValThrGluThrPro                                  275280285                                                                    TAAATTCTTAAAAATAAATGTTCCTTATGCCTCACTACCTCAAAAAAAAAAAAAAAAAAA931               AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA972                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 287 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ProLeuAlaPheLeuLeuGlnValTyrAlaProLeuProGlyArgAsp                              1 51015                                                                       AspAlaPheHisArgSerLeuPheLeuPheCysCysArgGluProLeu                              202530                                                                        CysCysAlaGlyLeuArgValP heArgAsnGlnLeuProArgLysAsn                             354045                                                                        AlaPheTyrSerTyrGluProProSerGluThrGlyAlaSerAspThr                              5055 60                                                                       GluCysValCysLeuGlnLeuLysSerGlyAlaHisLeuCysArgVal                              65707580                                                                      CysGlyCysLeuAlaProMetThrCysSerArgCysLysGlnAl aHis                             859095                                                                        TyrCysSerLysGluHisGlnThrLeuAspTrpGlnLeuGlyHisLys                              100105110                                                                     Gln AlaCysThrGlnSerAspHisLeuAspHisMetValProAspHis                             115120125                                                                     AsnLeuLeuPheProGluPheGluIleValThrGluThrGluAspGlu                              130 135140                                                                    IleGlyProGluValValGluMetGluAspTyrSerGluValIleGly                              145150155160                                                                  SerMetGluGlyValProGluGluG luLeuAspSerMetAlaLysHis                             165170175                                                                     GluSerLysGluAspHisIlePheGlnLysPheLysSerLysIleAla                              180185 190                                                                    LeuGluProGluGlnIleLeuArgTyrGlyArgGlyIleLysProIle                              195200205                                                                     TrpIleSerGlyGluAsnIleProGlnGluLysAspIleProAs pCys                             210215220                                                                     SerCysGlyValLysArgIlePheGluPheGlnValMetProGlnLeu                              225230235240                                                                  LeuAsn HisLeuLysAlaAspArgLeuGlyThrSerValAspTrpGly                             245250255                                                                     IleLeuAlaValPheThrCysAlaGluSerCysSerLeuGlyIleGly                              2 60265270                                                                    PheThrGluGluPheValTrpLysGlnAspValThrGluThrPro                                 275280285                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 ( A) LENGTH: 878 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..636                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CCCCAGCTGGCACATGGCTGCTACCCATGCCCTCCACACA GGCGCAAC48                           ProGlnLeuAlaHisGlyCysTyrProCysProProHisArgArgAsn                              151015                                                                        CTGGTAGAGGAGGTGAACGGCACCTACATGAAGAAGT GCCTCTATCAC96                           LeuValGluGluValAsnGlyThrTyrMetLysLysCysLeuTyrHis                              202530                                                                        AAGATTCAACACCCCCTGTGCCCAGTCTTCAACCTTG GCTATGTGGTG144                          LysIleGlnHisProLeuCysProValPheAsnLeuGlyTyrValVal                              354045                                                                        CGAGAGTCAGGCCAGGACTTCCGCAGCCTTGCTGAGAAGG GTGGGGTG192                          ArgGluSerGlyGlnAspPheArgSerLeuAlaGluLysGlyGlyVal                              505560                                                                        GTTGGTATCACCATTGACTGGAAGTGTGATCTGGACTGGCACGTTC GG240                          ValGlyIleThrIleAspTrpLysCysAspLeuAspTrpHisValArg                              65707580                                                                      CACTGCAAACCCATCTACCAGTTCCACGGACTGTATGGGG AGAAGAAC288                          HisCysLysProIleTyrGlnPheHisGlyLeuTyrGlyGluLysAsn                              859095                                                                        CTGTCTCCAGGCTTCAACTTCAGATTTGCCAGGCATT TCGTGCAGAAT336                          LeuSerProGlyPheAsnPheArgPheAlaArgHisPheValGlnAsn                              100105110                                                                     GGGACAAACCGTCGCCACCTCTTCAAGGTGTTTGGGA TTCACTTTGAT384                          GlyThrAsnArgArgHisLeuPheLysValPheGlyIleHisPheAsp                              115120125                                                                     ATCCTTGTGGATGGCAAGGCTGGGAAGTTTGACATCATCC CTACTATG432                          IleLeuValAspGlyLysAlaGlyLysPheAspIleIleProThrMet                              130135140                                                                     ACTACTATCGGTTCTGGGATTGGCATCTTTGGAGTGGCCACAGTGC TT480                          ThrThrIleGlySerGlyIleGlyIlePheGlyValAlaThrValLeu                              145150155160                                                                  TGTGATCTCTTATTGCTCCACATCCTGCCTAAGAGGCACT ACTACAAG528                          CysAspLeuLeuLeuLeuHisIleLeuProLysArgHisTyrTyrLys                              165170175                                                                     CAGAAGAAGTTCAAATATGCCGAGGACATGGGGCCGG GAGAGGGTGAA576                          GlnLysLysPheLysTyrAlaGluAspMetGlyProGlyGluGlyGlu                              180185190                                                                     CATGACCCCGTGGCCACCAGCTCCACTCTGGGCCTGC AGGAGAACATG624                          HisAspProValAlaThrSerSerThrLeuGlyLeuGlnGluAsnMet                              195200205                                                                     AGGACCTCCTGACCTTAGTCTTGAGATCCGGACTTGACGCAGTGTGT GG673                         ArgThrSer                                                                     210                                                                           CTTCCGGCAAGGGCTGATGGCTTTGAGCCAGGGCAGAGGGCATTCCCAGAGGCTTTCCCT733               GCAAGGCAGACACCAGTGGCCCTCTGGTTCAGCATGAAGACAGGCAAGACTTTGGATTTC793               A TAGCTCTGGTTTCAGTTCCACATGTCCCTTCCTGAGGGATGCCTCCTCCAGTTTTCTCC853              AATTTGGGTTCATATGGCTGGGCCC878                                                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 211 amino acids                                                    (B) TYPE: amino acid                                                         (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ProGlnLeuAlaHisGlyCysTyrProCysProProHisArgArgAsn                              151015                                                                        Leu ValGluGluValAsnGlyThrTyrMetLysLysCysLeuTyrHis                             202530                                                                        LysIleGlnHisProLeuCysProValPheAsnLeuGlyTyrValVal                              35 4045                                                                       ArgGluSerGlyGlnAspPheArgSerLeuAlaGluLysGlyGlyVal                              505560                                                                        ValGlyIleThrIleAspTrpLysCysAspL euAspTrpHisValArg                             65707580                                                                      HisCysLysProIleTyrGlnPheHisGlyLeuTyrGlyGluLysAsn                              8590 95                                                                       LeuSerProGlyPheAsnPheArgPheAlaArgHisPheValGlnAsn                              100105110                                                                     GlyThrAsnArgArgHisLeuPheLysValPheGlyIleHi sPheAsp                             115120125                                                                     IleLeuValAspGlyLysAlaGlyLysPheAspIleIleProThrMet                              130135140                                                                     ThrThrIleGly SerGlyIleGlyIlePheGlyValAlaThrValLeu                             145150155160                                                                  CysAspLeuLeuLeuLeuHisIleLeuProLysArgHisTyrTyrLys                              1 65170175                                                                    GlnLysLysPheLysTyrAlaGluAspMetGlyProGlyGluGlyGlu                              180185190                                                                     HisAspProValAlaThrSerS erThrLeuGlyLeuGlnGluAsnMet                             195200205                                                                     ArgThrSer                                                                     210                                                                           __________________________________________________________________________

What is claimed is:
 1. A purified and isolated DNA sequence andfragments and derivatives of the DNA sequence encoding a polypeptideeliciting programmed mammalian cell death, wherein the DNA sequence isset out in FIG. 5 (SEQ ID NO:1) .
 2. A purified and isolated DNAsequence and fragments and derivatives of the DNA sequence encoding apolypeptide eliciting programmed mammalian cell death, wherein the DNAsequence is set out in FIG. 6 (SEQ ID NO:3).
 3. A procaryotic oreucaryotic host cell transformed or transfected with DNA sequencesaccording to claim 1 with a heterologous regulatory control sequence inan expression vector therefor.
 4. A procaryotic or eucaryotic host celltransformed or transfected with DNA sequences according to claim 2 witha heterologous regulatory control sequence in an expression vectortherefor.
 5. A method of detecting the RP-8 gene encoding a polypeptideeliciting programmed mammalian cell death, comprising the stepsof:hybridizing mRNA extracted from a tissue or an organ sample with aprobe specific for the gene; and determining the ability of the probe tohybridize to the mRNA, wherein hybridization to the mRNA indicates thepresence of programmed mammalian cell death.
 6. A method of detectingthe RP-2 gene encoding a polypeptide eliciting programmed mammalian celldeath, comprising the steps of:hybridizing mRNA extracted from a tissueor an organ sample with a probe specific for the gene; and determiningthe ability of the probe to hybridize to the mRNA, wherein hybridizationto the mRNA indicates the presence of programmed mammalian cell death.7. The method of claim 5, wherein the tissue or the organ sample isselected from the group consisting of thymus, thymocytes, brain, liver,and olfactory epithelium.
 8. The method of claim 6, wherein the tissueor the organ sample is selected from the group consisting of thymus,thymocytes, brain, liver, and olfactory epithelium.